Project Summary: Mitochondria are organelles within the cell that have a variety of functions essential to maintaining overall cellular health. One of the unique features of this organelle is that it possesses its own DNA. Damage to mitochondrial DNA is linked to a broad range of biological and disease processes including aging, neurological dysfunction and cancer progression. Despite the critical nature of mitochondrial DNA, our knowledge of the basic mechanisms by which mitochondria repair damage to their DNA is limited. Much of what is known is restricted to a single type of DNA damage, oxidative lesions. This lack of progress in the mitochondrial DNA repair field results primarily from a lack of tools and techniques for identifying and characterizing mitochondrial DNA repair proteins. Recently, our lab developed a set of chemical probes that can induce a range of DNA lesions specifically in the mitochondrial genome. This approach is based on the retargeting of a number of known, and well-characterized, DNA-damaging agents specifically to mitochondrial DNA. Each of these retargeted compounds affect the mitochondrial DNA in a distinct manner (oxidative lesions, alkylation lesions, and double stranded breaks) allowing us to expand our understanding of individual cellular responses to a single type of DNA lesion. We hypothesize that by using this molecular toolbox we will be able to develop a comprehensive profile of mtDNA repair and elucidate novel responses for distinct lesion types within mitochondrial nucleic acids. In Aim 1 we will investigate the potential role of known nuclear DNA repair factors in response to a number of distinct types of DNA lesions within mitochondrial DNA. In Aim 2 we will broaden the search and look genome-wide to identify novel mitochondrial DNA damage repair proteins that are not linked to maintenance and repair of nuclear DNA. In Aim 3, we will characterize the biochemical roles of these proteins within the context of mitochondrial DNA repair. At the conclusion of these studies, we will have elucidated novel aspects of mitochondrial DNA damage repair and response. This work will provide the mitochondrial DNA damage community with a demonstration of the utility of mitochondria-targeted DNA damaging agents as functional probes of mitochondrial biology and generate a rich resource of functional screening data that can be used to spark new and exciting directions in mitochondrial DNA damage research. The proposed study will be the first to use high-throughput screening approaches coupled with highly specific chemical probes to study mitochondrial biology. We believe that this combination of approaches will provide new, important information about the function of an organelle that is critical for the supporting cellular life as well as regulating cellular death.